Sedimentation field flow fractionation is a versatile technique for the high resolution separation of a wide variety of particulates suspended in a fluid medium. The particulates include macromolecules in the 10.sup.5 to the 10.sup.13 molecular weight (0.001 to 1 .mu.m) range, colloids, particles, micelles, organelles and the like. The technique is more explicitly described in U.S. Pat. No. 3,449,938, issued June 17, 1969 to John C. Giddings and U.S. Pat. No. 3,523,610, issued Aug. 11, 1970 to Edward M. Purcell and Howard C. Berg.
Field flow fractionation is the result of the differential migration rate of sample components in a carrier or mobile phase in a manner similar to that experienced in chromatography. However, in field flow fractionation there is no separate stationary phase as there is in the case of chromatography. Sample retention is caused by the redistribution of sample components between the fast and the slow moving strata within the mobile phase. Thus, particulates elute more slowly than the solvent front.
Typically a field flow fractionation channel consists of two closely spaced parallel surfaces. A mobile phase is caused to flow continuously through the gap between the surfaces. Because of the narrowness of this gap or channel (typically 0.025 centimeters (cm)) the mobile phase flow is laminar with a characteristic parabolic velocity profile. The flow velocity is the highest at the middle of the channel and essentially zero near the two channel surfaces. An external force field of some type (the force fields include gravitational, thermal, electrical, fluid cross flow and others described variously by Giddings and Berg and Purcell), is applied transversely (perpendicular) to the channel surfaces or walls. This force field pushes the sample components in the direction of the slower moving strata near the outer wall. The buildup of sample concentration near the wall, however, is resisted by the normal diffusion of the particulates in a direction opposite to the force field. This results in a dynamic layer of component particles, each component with an exponential--concentration profile. The extent of retention is determined by the particulates time average position within the concentration profile which position is a function of the balance between the applied field strength and the opposing tendency of particles to diffuse.
In the case of a sedimentation force field, which is used in sedimentation field flow fractionation (SFFF), use is made of a centrifuge. A thin annular belt-like channel is made to rotate about the axis of the annulus. The resultant centrifugal force causes sample components of higher density than the mobile phase to sediment toward the outer wall of the channel. For equal particle density, because of their higher diffusion rate, smaller particulates will accumulate into a thicker layer against the outer wall than will larger particulates. On the average, therefore, larger particulates are forced closer to the outer wall.
If now the mobile phase or solvent is fed continuously from one end of the channel, it carries the sample components through the channel for later detection at the outlet of the channel. Because of the shape of the laminar velocity profile within the channel and the placement of particulates in that profile, solvent flow causes smaller particulates to elute first, followed by a continuous elution of components in the order of ascending particulate mass.
There are many criteria that a channel should meet in order to reduce the separation times required using this technique. One such criteria is that the channel must be relatively thin. Unfortunately, this creates many problems in that the walls of the channel should have a microscopically smooth finish to prevent the particles from sticking to the walls or being trapped in wall crevices. To provide such a microfinish, it is desirable to have access to the interior of the channel. Further, one must have access to the inner walls of the channel on occasion for cleaning. In order to maintain a high degree of resolution of the separated components of the sample, the thickness of the channel should be maintained constant during centrifugation. Constant channel thickness is difficult to maintain during centrifugation because the outer channel wall tends to enlarge to a greater extent then the inner channel wall. This is particularly true when the channel is formed between mating inner and outer rings. This is not easily accomplished, particularly if the weight of the channel elements are to be maintained at reasonably small values as is desired in centrifugation.
Grant, in his application, describes a channel construction which overcomes many of these disadvantages. The Grant channel is formed in a long, thin annular belt-like configuration. The channel is designed to maintain its thickness dimension constant and yet facilitate its manufacturing and cleaning by forming the channel of double mating rings in which the inner ring is split. This permits the inner ring to conform to and follow centrifugally induced expansions of the outer load carrying ring. The subject invention offers an alternative approach to that taught by Grant.